2.1. ENTEROTOXIGENIC BACTERIA AND DIARRHEAL DISEASE
Acute diarrheal disease due to the temporary colonization of the small intestine by enterotoxigenic strains of certain bacteria is a major health problem of global significance. Among the responsible bacteria, perhaps the most widely recognized is Vibrio cholerae. Less well known but of greater practical significance are particular strains of Escherichia coli (E. coli) which, together with rotavirus, produce acute diarrheic episodes that are fatal each year to an estimated 10 million infants living in underdeveloped tropical countries [Black et al., Lancet i: 141 (1981)].
These E. coli strains also generally account for a high incidence of the acute diarrhea that afflicts visitors to tropical regions. Furthermore, they have a profound impact upon livestock as well, since Kohler [J. Am. Vet. Med. Assoc. 173: 588 (1978)] has reported that weanling animals, particularly calves, lambs and piglets, may be similarly affected.
Both Vibrio cholerae and the enterotoxigenic E. coli strains produce their diarrheic effects through production of an enterotoxin. The cholera enterotoxin has been isolated and purified to homogeneity by Finkelstein [Crit. Rev. Microbiol. 2: 553 (1973)]. Furthermore, Finkelstein and LoSpalluto [J. Exp. Med. 130: 185 (1969)] have separated a protein subunit from the cholera toxin that has reduced biological activity. What has emerged from these and from other studies is the finding that the cholera enterotoxin is an 84,000 dalton protein that consists of an A and a B subunit.
The A subunit (28,000 daltons) is responsible for the biological effects of the toxin but is incapable of binding to its target receptors alone. Through the action of sulfhydryl reagents, the A subunit may be cleaved into two polypeptide chains, with molecular sizes of 7,000 and 21,000 daltons. Of these chains only the larger, designated A.sub.1, is active.
The B subunit, which has a size of 56,000 daltons, is essential for the expression of the activity of the A subunit. Apparently it acts by binding to a target cell and then facilitating penetration by the active A subunit. Finkelstein et al. J. Immunol. 113: 145 (1974)] have that the B subunit consists of non-covalently associated subunits that can be dissociated by vigorous treatment with sodium dodecyl sulfate or at low pH with urea into five polypeptide chains.
The effects of cholera toxin have been demonstrated by Sheer et al. Gastroenterology 65: 895 (1973)] in rabbit jejunum. In that system, the toxin causes a blood to lumen unidirectional flux of sodium. As a result, the intestinal fluid becomes low in protein, Mg.sup.++ and high in K.sup.+, Na.sup.+ and HCO.sub.3.sup.-, compared to normal serum levels. With these ionic changes, there is a concomitant outflowing of water to the lumen, for the maintenance of osmotic equilibrium with the blood plasma.
The precise structure of the cholera toxin receptor is unknown, but it appears to be a glycolipid. This observation is based upon a finding by King and van Heyningen [J. Infect. Dis. 131: 643 (1975)] that the binding of cholera toxin to membranes is inhibited by various glycosphingolipids. Of the compounds of this type examined, G.sub.M1 (galactosyl-N-acetylgalactosaminyl-examined, (sialyl)-galactosylglucosylceramide) was most potent.
Once cholera toxin binding occurs, there is a stimulation of adenylate cyclase activity and a locking of that enzyme in the activated state. The result is an increase in intracellular levels of cAMP that in some way gives rise to the above ionic changes.
Enterotoxic strains of E. coli also mediate their diarrheic effects through the production of enterotoxins. These toxins are of two types, one of which is a relatively low molecular weight species of 2,000 daltons. Because it survives treatment at 100.degree. C., this species is referred to as the heat-stable toxin (ST). A second toxin that is heat labile (LT) is remarkably similar to the cholera toxin.
As shown by Gill et al. [Infect. Immun. 33: 677 (1981)], E. coli LT consists of the same type and number of subunits as the cholera toxin, and the corresponding subunits have approximately the same molecular weights. As with cholera toxin, the B subunits of LT attach to intestinal mucosal glycolipid receptors, thus permitting penetration of the cell by the biologically active A subunit. The sequence of events from that point on is also similar. Most importantly, Clements and Finkelstein [Infect. Immun. 21: 1036 (1978)] have shown that E. coli LT is immunologically related to both the A and B subunits of cholera enterotoxin.